NFKB induction - to test promoter activity (Sep/14/2007 )
I'm testing the activity of a promoter, I did PCR and started with a 2000bp fragment using pCAT vector, I did bioinformatic analysis and I want to study if it's regulated by NFKB TF, as I don't have plasmids with nfkb expression, neither have lots of money to spend, I' m planning to induce nfkb activation and to test its effect on the reporter expression.....I have some questions about this and it would be great if any of you can give me some answers....
First of all, how do I induce nfkb? and how do I know that if there is an effect on activity, then it's because of nfkb and not because of other factor stimulated by the method I used? I know it can be done with LPS or IF, but this factors affect exclusively nfkb?...
.....how this things work, does anyone know?? (I mean LPS and IF), is there anything specific to promote IKK and NFKB unbinding (I know NFKB is in cytoplasm associated with its inhibitor and when needed it moves to the nucleus, after being released by its inhibitor)....
.....something else, when doing transfection, the plasmid stays in cytoplasm, right? (or enters de nucleus?) and if that's the case, NFKB recognizes the specific sequences in the promoter region in the reporter vector, in the cytoplasm?? is not necessary for it to be in the nucleus enviroment??
hope you can help me and make me wiser!!
Multiple signaling pathways converge to the activation of NF-KB. You need to read the background info yourself to appreciate how complicated it is.
I heard someone joked once like this: you may accidentally activate NK-KB if you wisp to your cells.
I do want to tell yout that most transfection experiments’ are injurious to cells and the procedure itself can activate NF-Kb. So be careful on this.